Abstract

We and others have
proposed mammalian cells as gene delivery vehicles with the potential
for overcoming physiological barriers to viral vectors. To that end, we
previously have shown the potential of CD34+ endothelial
progenitors for systemic gene delivery in a primate angiogenesis model.
Here we seek to explore the utility of CD34+ cells of human
origin as vehicles for toxin genes and, in particular, to measure their
capacity to effect a cytotoxic bystander effect in human endothelium
and tumor cells. To this end, CD34+ cells were transduced
with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine
kinase. To test the capacity of CD34+ cells to
induce a cytotoxic bystander effect in target cells, we performed
mixing experiments, whereby TOZ.1-transduced CD34+ cells
were mixed with either human vascular endothelial cells or human
ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the
MTS assay. Lastly, mixtures of TOZ.1-transduced
CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to
evaluate the bystander effect in vivo. After
transduction of CD34+ cells with TOZ.1, treatment with
ganciclovir induced the killing of 99% of cells. In cell-mixing
experiments, a linear correlation was observed between the percentages
of TOZ.1-transduced CD34+ cells and total cell killing. For
example, when 50% of CD34+ transduced cells were mixed
with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when
the same percentage was mixed with human vascular endothelial cells,>
80% of the total number of cells died. In vivo
studies showed an abrogation of tumor formation when TOZ.1-transduced
CD34+ cells and ganciclovir were administered. Our
observations establish the feasibility of a method for cell-based toxin
gene delivery into disseminated areas of tumor angiogenesis.

INTRODUCTION

A number of strategies have been developed to accomplish cancer
gene therapy. One of these is molecular chemotherapy. Although similar
in principle to conventional chemotherapeutic interventions for cancer,
molecular chemotherapy is designed to achieve selective expression of a
toxin gene in the tumor site by transferring the corresponding DNA.
Typically, the approach involves both the transfer into the tumor cells
of a gene encoding an enzyme and the administration to the patient of
an inactive, innocuous prodrug. When the delivered enzyme is expressed,
it transforms the prodrug into a cytotoxic metabolite. Purportedly this
happens exclusively within the genetically modified tumor cell, thus
establishing the basis for a favorable therapeutic index and for a
definite advantage with respect to conventional chemotherapy
(1)
.

Fundamental to the clinical implementation of this strategy in patients
with advanced cancer is the development of a vector system that allows
direct, extensive, and efficient in vivo gene transfer into
disseminated tumors. In this regard, both viral and nonviral vectors
previously have been used clinically for the treatment of cancer,
mostly in a loco-regional context. Among the vector systems that have
been used successfully are viruses (retrovirus, adenovirus, and
adeno-associated virus, poxvirus, herpesvirus), naked DNA, and lipids.
In general, despite their utility in selected applications, these
vectors have been limited in their ability to accomplish highly
efficient gene delivery to target cells in clinically relevant models
(1)
. Furthermore, for disseminated cancer, vectors with
consistent capacities for systemic gene delivery have not been
available (2)
. Thus, shortcomings in the present
generation of vectors have limited the overall efficacy and
implementation of gene-based molecular chemotherapy for advanced
cancer.

As an alternative to these aforementioned vector approaches, cells have
also been used as vectors. In this approach, cells are removed from the
body, and therapeutic genes are transferred to the cells
extracorporally, followed by their re-implantation back into the
patient. In this manner, the genetically modified cell itself becomes
the ultimate vector for gene delivery. For example, the pioneering
clinical use of primary cells as so-called “cellular vehicles” is
based on T lymphocytes (3, 4, 5, 6)
. Conceivably, a variety of
genes can be introduced in candidate cellular vehicles, and expression
of these payloads can be obtained in an environment where these cells
localize. A loco-regional effect subsequent to the expression of the
therapeutic gene can thus be achieved in areas otherwise inaccessible
to direct gene transfer. In this regard, our group has shown that
mature human endothelial cells can be used in vivo in a
murine model as cellular vehicles to deliver a toxin gene regionally
and induce cytotoxicity in i.p. ovarian cancer cells (7)
.

For the clinical application of cellular vehicles in the context of
disseminated malignant disease, however, a cellular vector should have
the attributes of systemic distribution, adequate tumor tropism, ready
availability, and lack of immunogenicity. Interestingly, a novel role
has been described recently for stem cells, known as
EPCs,2
obtained
from the bone marrow, peripheral circulation, or cord blood
(8, 9, 10)
. Phenotypically, these cells are characterized by
the expression of the cellular surface markers CD34 and KDR/Flk-1
(8, 9)
, which is a receptor for vascular endothelial
growth factor. Another possible marker described recently is AC133
(11)
. A very intriguing aspect of the EPCs’
behavior, originally described in animal models of limb ischemia, is
their capacity to localize into areas of angiogenesis after their
systemic administration (8)
. Thus, endothelial progenitors
might be further developed as a novel cellular vector with unique
features, based on their capacity for systemic circulation and natural
tropism to areas of active angiogenesis. Previously, we have
demonstrated the ability of genetically modified
CD34+ cells to localize into areas of
angiogenesis in a nonhuman primate skin model (12)
.
Furthermore, in rhesus animals infused with autologous
CD34+ EPCs transduced with a TK-encoding herpes
virus, skin autografts underwent necrosis or accelerated regression
after administration of GCV (12)
. Thus, EPCs not only
localize into areas of angiogenesis but are also able to express a
therapeutic gene and induce therein a meaningful biological effect.

In this report, we further explore the capacity of human
CD34+ EPCs to serve as vehicles for toxin gene
delivery into the tumor vasculature and their capacity to exert an
effective cytotoxic bystander effect over human tumor and primary
endothelial cells.

Recombinant Herpes Simplex Vectors and Cell Infection.

For genetic modification of CD34+ cells, we used
the nonreplicative herpes virus vector TOZ.1, which is deleted of the
genes ICP4, ICP22, ICP27, and
UL41, and encodes the reporter gene lacZ.
As a control, we used a similar vector lacking the lacZ
gene, T3. Both vectors encode the HSV-TK gene. The
construction and preparation of these vectors, derived from a KOS
strain mutant, has been described elsewhere (14)
. Viral
stocks were purified by centrifugation in OptiPrep (Life Technologies,
Inc.), and titered using 7B cells, which express both the
ICP4 and ICP27 genes.

Before infection, cells were washed twice with PBS and resuspended
within microcentrifuge tubes with appropriate cell-specific media. The
suspended cells were incubated with HSV vectors using MOI doses of 0.3,
3, and 10 plaque-forming units, with gentle rocking for 2 h at
37°C.

Aliquots of transduced cells or peripheral blood mononuclear cells were
stained with antihuman CD34+-biotin antibody
(Cellpro, Bothell, WA) or an irrelevant antibody conjugated with
streptavidin-PerCP (Becton Dickinson, San Jose, CA). After 20 min of
incubation in the dark, the cells were washed with PBS and then stained
with FDG (Sigma, St. Louis, MO) by the FACS-gal technique of Fiering
et al.(15)
. Briefly, after incubation
in a 37°C water bath for 5 min, 50 μl of distilled water containing
2 mm FDG were mixed thoroughly with an equal
volume of cells suspended in staining medium [10
mm HEPES, and 4% FCS in 1× PBS (pH 7.3)]. The
cells were then returned to the water bath for exactly 1 min. FDG cell
loading was stopped by adding 500 μl of ice-cold staining medium. The
cells were maintained on ice until FACS analysis was performed within
the following 30 min. Cells not incubated with FDG served as the
negative control. Flow cytometry data acquisition was performed using a
FACS caliber flow cytometer (Becton Dickinson, Mountain View, CA), and
analysis was supported by Cell Quest software.

In Vitro Analysis of the Cytotoxic Bystander Effect.

Cell mixing experiments were performed to assess for a bystander
effect, as described previously (16)
. Briefly,
CD34+ cells were transduced with the recombinant
herpes virus TOZ.1 and then mixed at different ratios with
non-HSV-infected cells (SKOV3.ip1 and HUVECs). As a control,
TOZ.1-transduced SKOV3.ip1 cells were mixed with nontransduced
SKOV3.ip1. The ratios of TOZ.1-infected cells to uninfected cells were
as follows: 0:100, 10:90, 50:50, 90:10, and 100:0. Cell mixtures were
plated in triplicate in 96-well plates at a total density of 20,000
cells/well. Twenty-four h later, half of the samples were treated with
GCV (20 mm). Cell viability was determined 6 days later by
a colorimetric cell proliferation assay measuring the conversion of a
tetrazolium salt to formazan by viable cells, as described by the
manufacturer (MTS assay; Cell Titer 96 Aqueous Nonradioactive Cell
Proliferation Assay; Promega, Madison, WI). To this end, the
plates were incubated for 4 h at 37°C after the addition of 20μ
l of assay solution. Subsequently, the absorbance was measured at
490 nm by an ELISA reader (Molecular Devices, Menlo Park, CA). To
quantify the cell numbers, a standard curve was created by plating
nontreated cells in triplicate wells at the following concentrations:
0; 2,000; 10,000; 20,000; and 50,000 cells/well. The total numbers of
cells in the experimental groups were calculated based on the standard
curve, using SOFT max computer software (Molecular Devices).

In Vivo Analysis of the Cytotoxic Bystander Effect.

To study the ability of the cytotoxic bystander effect to inhibit tumor
growth, female nude mice (ages 6–8 weeks; obtained from the National
Cancer Program, Frederic, MD) were treated as follows. Animals
(n = 24) received injections in each flank of a mixture
of tumor and CD34+ cells (four s.c.
injections/mouse), including (a)
CD34+/TK+
(CD34+ cells transduced with HSV-TK) mixed with
ovarian cancer SKOV3.ip1 cells; (b)
CD34+/TK− (nontransduced)
mixed with SKOV3.ip1 cells; or (c) SKOV3.ip1 alone. Each
animal received a total of 2.5 × 106 tumor
cells/nodule. The CD34+ cells had been infected
in vitro with the recombinant herpes virus TOZ.1 at a MOI of
10 plaque-forming units. Two h post infection, the cells were washed
twice and resuspended with PBS. The infected
CD34+ cells were then mixed with SKOV3.ip1 cells
at a 1:1 ratio (2.5 × 106
CD34+ and 2.5 × 106
SKOV3.ip1 cells), and injected s.c. into the
CD34+/TK+ and
CD34+/TK− groups.

Twenty-four h after injection of the cellular mixture, half of the
animals were started on a treatment of daily i.p. injections of GCV (50
mg/kg) for 10 days. Tumor volumes were measured every 3 days during
treatment for 1 month, using the formula: volume = (width ×
height × diameter)/2. Animal experiments were performed according
to institutional guidelines and under the supervision of the Animal
Resources Program at the University of Alabama at Birmingham.

RESULTS

Our molecular chemotherapy approach is predicated upon the
capacity of the proposed cellular vehicles to induce cytotoxicity,
mediated by the TK gene, within angiogenic vessels and in
the immediate vicinity of surrounding tumor deposits. Testing this
feature amounts to determining the potency of the toxin gene as
delivered by the cellular vehicle on bystander human endothelial and
tumor cells.

A Recombinant HSV Vector Efficiently Transduces Human CD34 Cells.

To determine the efficiency of transduction of human
CD34+ cells by a herpes virus vector,
CD34+ cells were incubated with various MOI doses
(0, 0.3, 3, or 10) of TOZ.1, a nonreplicative, recombinant
lacZ gene-encoding HSV. Twelve h later, FACS analysis was
carried out using FDG staining. More than 95% of cells were positive
in this analysis with a MOI of 3 (Fig. 1⇓A). Fewer than 1% of cells
were positive when infected with a control HSV vector. Additionally,
TOZ.1-infected cells developed an intense blue pigmentation after
staining with X-gal, confirming that CD34+ cells
can be efficiently transduced with a herpes virus vector (data not
shown).

A, transduction efficiency of
TOZ.1 in human CD34+ cells. Highly enriched
CD34+ cells were plated in 1 ml of serum-free StemPro
culture medium. Cells were incubated at 1 × 106
cells/well in a 12-well flat-bottomed plate. Herpes virus stock was
added directly to the cultures at MOI doses of 0, 0.3, and 3.
Infections were initiated 1 h after cultures were started.
Staining for LacZ was performed after 48 h, by which time, 99% of
the CD34+ cells had been transduced and expressed the blue
color. B, toxin gene killing demonstrated in
HSV-TK-transduced CD34+ cells at 3 MOI. Transduced cells
(20,000) were plated in quadruplicate in 96-well plates. Half of the
samples were treated with 20 μm GCV (+GCV;□
). Five days later, the percentage of viable cells was measured
using a MTS assay. Bars, SD.

TOZ.1-transduced CD34+ Cells Are Susceptible to the
Cytotoxic Effect of GCV.

We next investigated the utility of TOZ.1, which also encodes TK, to
achieve toxin gene expression in CD34+ cells and
to produce cytotoxicity after GCV treatment. For this analysis,
TOZ.1-transduced cells (MOI of 10) were treated with the prodrug GCV
(20 μm). Four days later, viable cells were stained using
a MTS assay. The experiment demonstrated that GCV readily killed the
HSV-TK-infected cells. Infected cells not exposed to GCV remained
viable (Fig. 1⇓B). Of note, GCV had no demonstrable toxic
effect on uninfected CD34+ cells at the dose
tested (data not shown).

Fundamental to the efficiency of the molecular chemotherapy strategy is
the bystander effect, whereby toxin gene-expressing cells exert a
noxious effect on surrounding untransduced cells. We, thus,
investigated whether the HSV-TK-expressing CD34+
cells could functionally accomplish a bystander effect with a series of
human ovarian carcinoma cell lines and with human endothelial cells
(HUVECs) in vitro. A mixing experiment was performed in
which TOZ.1-transduced CD34+ cells were mixed at
various ratios with nontransduced CD34+ cells,
human ovarian cancer cells (SKOV3.ip1), and HUVECs. GCV-mediated
killing was measured using a MTS assay. Studies were performed with
CD34+ cells to determine whether they could
induce bystander cytotoxicity into homologous, nonadherent cells. In
effect, as few as 10% of HSV-TK-transduced CD34+
cells achieved killing of ∼60% of the total population of
CD34+ cells (Fig. 2⇓A). A similar effect was
observed when CD34+ cells were mixed with ovarian
cancer SKOV3.ip1 cells (Fig. 2⇓B). Lastly, when 50% of
HSV-TK-transduced CD34+ cells were mixed with
HUVECs, >80% of the total number of cells died (Fig. 2⇓C).
Of note, CD34+ cells remained in the dish mostly
as unattached, floating cells, which restricted the contact of
HSV-TK-transduced CD34+ cells over other target
cells. Interestingly, the highest levels of bystander effect were
observed when the cell mixtures were incubated with gentle rocking,
suggesting the importance of intercellular contact between the
suspended CD34+ cells and the other cell types.

Having established that CD34+ cells are an
effective vehicle for delivery of cytotoxicity to endothelial and tumor
cells in vitro, we next evaluated this cellular
vehicle-based approach for therapeutic potential in an in
vivo model of human carcinoma of the ovary. For this analysis,
nude mice received s.c. xenografts of SKOV3.ip1 cells and
CD34+ cells infected with TOZ.1 and mixed at a
ratio of 1:1, as described above. The control groups consisted of
animals receiving either SKOV3.ip1 cells alone or nontransduced
CD34+ and SKOV3.ip1 cells in a 50:50 ratio. Half
of the animals received daily i.p. injections of GCV (50 mg/kg) for 10
consecutive days. The animals that had received HSV-TK-transduced
CD34+ and SKOV3.ip1 cells and were treated with
GCV showed no tumor nodules. In contrast, all animals in the control
groups developed large tumor nodules at day 28 (Fig. 3⇓A). After 6 weeks of
observation, nodules were still visible in animals not receiving GCV
and in those treated with nonmodified CD34+ cells
(Fig. 3⇓B). However, no nodules were detected in the animals
that received HSV-TK-transduced CD34+ cells and
treatment with GCV (Fig. 3⇓B). Thus, genetically
modified human CD34+ cells can effectively induce
a toxic bystander effect in murine endothelium and human cancer cells
in vivo.

In vivo bystander effect.
HSV-TK-transduced CD34+ cells or nontransduced cells were
mixed at a ratio of 50:50 with ovarian carcinoma SKOV3.ip1 cells. A
total of 5 × 106 cells (2.5 × 106
CD34+ plus 2.5 × 106 SKOV3i.p1) were
injected into each flank of nude mice (n = 4; four
nodules/mice). Half of the animals were treated with GCV (50 mg/kg) for
10 days. Tumor volume was measured every 3 days. The experiment was
repeated, with one set selected as representative. A,
evolution of tumor volume during a monitoring period of up to 6 weeks.
B, the photos show a representative animal from each
group, 4 weeks after treatment.

DISCUSSION

Our ultimate goal is to design a viable vector system that allows
systemic gene delivery into multiple areas of tumor angiogenesis. To
this end, we report here on our further attempts to develop an
autologous cellular vehicle based on human EPCs, which purportedly
localize into areas of angiogenesis. A basic requirement for our
proposed cellular system is the ability to determine an antitumoral
effect by affecting cells in the vicinity via expression of an
exogenous therapeutic gene, i.e., to induce a bystander
effect.

As a first iteration, we explored the utility of human
CD34+ cells genetically modified with the
TK gene from HSV. The use of human
CD34+ cells, which are typically hard to
transduce at high levels with the vectors available at present, places
a high level of stringency on this approach, which we wanted given our
orientation toward therapeutic applicability. In this regard, we
consider the use of autologous cells a potentially critical advantage
of our strategy to assure safety and a long enough survival of the cell
vehicles (17)
. The other determinant of the efficacy of
our proposed utilization of cellular vehicles for inducing tumor
regression is the potency for exerting cytotoxicity in surrounding
tumor cells, host cells, and the stroma, including the vasculature.
Indeed, we have found that the genetically modified cells themselves
are sensitive to the toxic effect of GCV and that they exert a
bystander cytotoxic effect on both adjacent tumor cells and human
endothelium. Using the strategy of cell-based induction of
cytotoxicity, we have accomplished a killing effect on tumor cells
comparable to that obtained by direct viral vector-mediated toxin gene
delivery. In addition, we have observed a comparable toxin sensitivity
in primary human endothelial cells, which suggests that the bystander
effect similarly applies to both cell types, arguably the key targets
for our cytotoxic intervention. Thus, we have advanced the evaluation
of our system as regards to the capacities of the proposed cellular
substrate, i.e., human CD34+ cells.

In our previous studies, we established the initial
proof-of-concept with respect to the strategy of using circulating EPCs
as cellular vehicles for systemic gene delivery (12)
. The
initial goal was to develop a vehicle able to systemically reach
multiple disseminated areas of angiogenesis. To this end, we sought to
exploit the natural tropism of cells recently described as EPCs and
existent within the CD34+ population of
mononuclear cells from mobilized peripheral blood (8, 9)
.
In this regard, multiple studies in embryos and a variety of
transplantation studies have supported the existence of EPCs,
i.e., cells localized in the bone marrow that would have the
capacity to migrate, proliferate, and differentiate into mature
endothelial cells. Additional recent evidence suggests that these cells
can be characterized by the expression of the immunophenotypic marker
AC133 within the CD34+,
FLK-1+ population (11)
. Although we
did not select any subpopulation of cells among those positive for
CD34, the obtainment of very high levels of gene transfer argues for
the genetic modification of a significant fraction from the general
population. Our previous results in the rhesus model support this
hypothesis. Eventually, the optimal cell subpopulation, endowed with a
more specific angiogenic tropism behavior, will be selected by flow
cytometry, genetically modified, and used for systemic gene delivery.
Thus, we contend that ultimately, a defined subset of circulating human
cells can be conveniently exploitable as “Trojan horses” targeted
into areas of angiogenesis.

A requisite property of our proposed cellular vehicle in its current
TK gene configuration, however, is to exert a potent enough
cytotoxic bystander effect. Although our results in the rhesus model
suggest that such an effect occurred, as did the dramatic toxic effect
induced in skin grafts with only a limited number of modified EPCs
detected in situ, we sought here to formally examine this
hypothesis. We have here demonstrated that the transduced
CD34+ cells express the TK gene with
high efficiency and, thus, become sensitive to the effect of the
prodrug GCV, with ensuing quantitative cell killing. Moreover, we have
confirmed that a cytotoxic bystander effect can be induced by
genetically modified CD34+ cells in juxtaposition
to tumor cells. Furthermore, we have demonstrated that this bystander
effect is also achieved against proliferating human primary endothelial
cells. More importantly, tumorigenicity was inhibited in
vivo as a consequence of the bystander cytotoxicity. These
augmented effects might thus potentially overcome the requirement to
achieve near-universal quantitative tumor cell transduction in tumor
foci, a major challenge in gene therapy for cancer. In effect, the
establishment of a bystander effect has been an important factor
contributing to the overall efficiency of molecular chemotherapy
approaches in gene therapy (1)
. In this regard, numerous
studies have examined the biological basis of the bystander effect.

It appears that the presence of intercellular gap junction
communications is an important determinant of the bystander phenomenon,
although not a universal one (18)
. In addition, apoptotic
bodies generated during killing by toxin gene delivery may function as
a vehicle for disbursement of toxin gene metabolites to nontransduced
tumor cells (19)
. In most cases, this effect has been
sought between tumor cells or between tumor cells and surrounding
endothelial cells (20)
. To our knowledge, we have herein
proposed for the first time the exploitation of a bystander effect
induced from genetically modified cells with endothelial lineage.

The use of circulating cellular vehicles was first proposed a decade
ago. Then, human TILs were used as vehicles for retroviral-mediated
gene transfer based on a putative preferential localization at tumor
sites (3, 4)
. It was also hypothesized that TILs could be
an enriched source of natural killer cells and CTLs specific for
tumor antigens. On this basis, technology for their expansion in
culture was developed, and TILs were the first immune cells to be
genetically modified and applied in a human gene therapy clinical trial
against cancer (21)
. It was soon observed that whereas
TILs do include CTL- and natural killer-activated cells, only a few of
these cells from these mixed cell populations are specific for the
tumor from which they are isolated. Furthermore, reinfused TILs
localize poorly into tumors, and their required expansion in
vivo using interleukin-2 was rather toxic to patients. Although
several strategies have been applied to improve treatments based on
TILs and other lymphocytes, including an elegant reengineering of their
tropism (22, 23)
, modest localization of TILs in tumors
remains a limitation for the efficacy of this poorly tolerated and
expensive therapy.

In contrast, the target of our proposed
CD34+-based cellular vehicles is the nascent
vascular endothelium of angiogenic tumors, which should be much more
accessible to these circulating cells. This hypothesis is being tested
in vivo. On the other hand, mature endothelial cells have
also been shown to localize into areas of angiogenesis
(24)
and have been used effectively to deliver
apolipoprotein E as a treatment for hypercholesterolemia in a
murine apolipoprotein E knockout atherosclerosis model
(25)
and for delivery of the TK toxin (7)
. In
both cases, the major limitation to further development of the strategy
is our inability to isolate and expand in vitro mature
autologous endothelial cells. Furthermore, the alternative use of
allogeneic cells established and expanded in vitro would
introduce all of the encumbrances of an allogeneic cellular transplant.
In marked contrast, we have proposed herein the use of autologous
CD34+ cells, which are easily obtainable in large
numbers from peripheral blood. Our use of these cells is in accord with
the expanding use of progenitor and stem cells in a variety of
therapeutic contexts (26, 27)
. The potential of
CD34+ cell subsets as novel systemic vectors for
toxin and other genes warrants further investigation. Our observations
to date clearly establish the feasibility of cell-based gene delivery
into disseminated areas of tumor angiogenesis as a rational strategy
for cancer gene therapy.

Acknowledgments

We thank Drs. Joanne T. Douglas, Masato Yamamoto, and Enrique
Casado for critical reading of the manuscript and helpful suggestions.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.